Patent application title: USE OF A CD28 BINDING SUBSTANCE FOR MAKING A PHARMACEUTICAL COMPOSITION

Abstract:

The invention relates to the use of a CD28-specific superagonistic
monoclonal antibody (mAb) or of a mimicry compound hereto for making a
pharmaceutical composition for the induction and/or multiplication of
regulatory T cells.

Claims:

1. A method for the treatment or prophylaxis of autoimmune diseases or
inflammatory reactions, wherein a pharmaceutical composition for the
induction or multiplication of regulatory T cells in vitro or in vivo.

2. The method of claim 1, wherein the composition is administered for the
treatment or prophylaxis of autoimmune diseases.

3. The method of claim 1, wherein the composition is administered for the
treatment of the Guillain-Barre syndrome (GBS) or of the chronic
demyelinating polyneuropathy (CDP).

4. The method of claim 1, wherein the mAb can be produced from a non-human
mammal is immunized with CD28 or a partial sequence thereof.

5. The method of claim 1, wherein the mimicry compound is obtainable by a
screening method, in which a prospective mimicry compound or a mixture of
prospective mimicry compounds is subjected to a binding assay with CD28
or a partial sequence thereof, wherein the CD28 partial sequence
comprises a C'D loop, and substances binding to CD28 or to the partial
sequence therefore are selected, followed by an assay for testing for
superagonistic stimulation of several to all sub-groups of T lymphocytes.

6. The method of claim 1, wherein the mAb is obtainable from hybridoma
cells, as filed under the DSM numbers DSM ACC2531 (mAb: 9D7 or 9D7G3H11)
or DSM ACC2530 (mAb: 5.11A or 5.11A1C2H3).

7. The method of claim 1, wherein the mAb or the mimicry compound
comprises one or more of the sequences Seq. ID 9, 11, 13 or 15, or one or
more of the sequences Seq. ID 10, 12, 14, 16 or one or more of the
sequences 18 or 19, or sequences being homologous thereto.

8. A method for the treatment or prophylaxis of autoimmune diseases or
inflammatory reactions, wherein eithera pharmaceutical composition is
administered to a patient, comprising a CD28-specific superagonistic
monoclonal antibody or a mimicry compound hereto and in a galenic
preparation for a defined and suitable form of administration comprising
intraveneous injection; ora body liquid is taken from a patient,
comprising blood comprising T lymphocytes or precursor cells hereto, and
the body liquid, possibly after a processing step, is reacted with a
CD28-specific superagonistic monoclonal antibody or a mimicry compound
hereto, and the thus treated body liquid is again administered to the
patient.

9. The method of claim 4, wherein the CD28 partial sequence comprises a
C'D loop, wherein cells are taken from the non-human mammal, hybridoma
cells are produced from the cells, and the thus obtained hybridoma cells
are selected such that in their culture supernatant there are mAbs that
superagonistically bind to CD28.

10. The method of claim 8, wherein the body liquid is administered to the
patient by intraveneous injection.

Description:

STATEMENT OF RELATED APPLICATIONS

[0001]This application is a continuation of U.S. patent application Ser.
No. 11/585,484, filed Oct. 24, 2006, entitled "Use of a CD28 Binding
Substance for Making a Pharmaceutical Composition", which is a
continuation of U.S. patent application Ser. No. 10/389,679, filed Mar.
13, 2003, entitled "Use of a CD28 Binding Substance for Making a
Pharmaceutical Composition," now abandoned. Both of the prior
applications are incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

[0002]The invention relates to the use of a CD28 binding substance for
making a pharmaceutical composition.

DEFINITIONS

[0003]Monoclonal antibodies (mAbs) are antibodies which are produced by
hybrid cell lines (so-called hybridomas) which typically have been
generated by fusion of a B cell of animal or human origin producing
antibodies with a suitable myeloma tumor cell.

[0004]The amino acid sequence of human CD28 is known under accession No.
NM--006139.

[0005]The C'-D loop of CD28 comprises the amino acids 52 to 66 of the
above CD28 sequence (for numbering see also Ostrov, D. A., et al.;
Science (2000), 290:816-819). The term C'-D loop will also include in the
following any partial sequences therefrom.

[0006]A loop or a binding site arranged therein is freely accessible, if
for a defined binding partner for the binding site in the loop there is
no steric hindrance by the sequences or molecules following to the loop.

[0007]Regulatory T cells are CD4+ T cells inhibiting in a mixture with
naive CD4+ T cells the activation thereof. Hereto belong in particular
CD4+CD25+ T cells. Another feature of regulatory T cells is, compared to
other T cells, a low expression of the high-molecular isoforms of CD45
(human: RA). For regulatory T cells, the constitutive expression of CD152
is typical. CD4+CD8-SP thymocytes are one of the essential sources for
regulatory T cells. For a further characterization of regulatory T cells,
reference is made to the document K. J. Maloy et al., Nature Immunology,
Vol. 2, No. 9, pages 816 ff., 2001.

[0008]The induction of regulatory T cells is the increase of the metabolic
activity, enlargement of the cell volume, synthesis of immunologically
important molecules and beginning of the cell division (proliferation)
upon an external stimulation. As a result, after the induction there are
more regulatory T cells than before.

[0009]Homology is an at least 70%, preferably at least 80%, most
preferably at least 90% sequence identity on a protein level, a
homologous protein or peptide binding a defined binding partner with at
least identical affinity. Deviations in the sequence may be deletions,
substitutions, insertions and elongations.

[0010]A mimicry compound is a natural or synthetic chemical structure
behaving in a defined binding assay as a defined mAb mimicrying the
mimicry compound.

[0011]The term mAbs comprises, in addition to structures of the
conventional Fab/Fc type, also structures exclusively consisting of the
Fab fragment. It is also possible to use the variable region only, the
fragment of the heavy chains being connected with the fragment of the
light chain in a suitable manner, for instance also by means of synthetic
bridge molecules, such that the binding regions of the chains form the
antibody epitope. The term antibody also comprises (possibly complete)
chimeric and humanized antibodies.

[0012]Superagonistic stimulation of the proliferation of CD28-specific
cells means that no costimulation, i.e. no further binding event in
addition to a binding of a mAb or of a mimicry compound to CD28 is
necessary for the stimulation or inhibition of the proliferation.

BACKGROUND OF THE INVENTION AND PRIOR ART

[0013]For understanding the invention, firstly the following technological
background is important. The activation of resting T cells for the
proliferation and functional differentiation firstly requires the
occupation of two surface structures, so-called receptors: 1. of the
antigen receptor having a different specificity from cell to cell and
being necessary for the detection of antigens, e.g. viral fission
products; and 2. the CD28 molecule expressed on all resting cells with
the exception of a sub-group of the human CD8 T cells, said CD28 molecule
naturally binding to ligands on the surface of other cells of the immune
system. This is called the costimulation of the antigen-specific immune
reaction by CD28. In a cell culture, these processes can be imitated by
occupation of the antigen receptor and of the CD28 molecule with suitable
mAbs. In the classic system of the costimulation, neither the occupation
of the antigen receptor nor that of the CD28 molecule alone will lead to
the T cell proliferation, the occupation of both receptors is however
effective. This observation has been made with T cells of man, mouse and
rat.

[0014]There are however also known CD28-specific mAbs that can initiate
the T cell proliferation without costimulation. Such a superagonistic,
i.e. independent from the occupation of the antigen receptor, activation
of resting T lymphocytes by CD28-specific mAbs is known in the art from
the document Tacke et al., Eur. J. Immunol., 1997, 27:239-247. According
thereto, two types of CD28-specific monoclonal antibodies having
different functional properties are described: costimulatory mAbs
costimulating the activation of resting T cells only with simultaneous
occupation of the antigen receptor; and superagonistic mAbs which can
activate T lymphocytes of all classes in vitro and in the test animal for
proliferation without occupation of the antigen receptor. Both in so far
known mAbs originate from an immunization with cells, on which rat CD28
is expressed, and are obtainable by different selections directed to
their respective properties.

[0015]From the document DE-197 22 888 it is known in the art that
superagonistic mAbs are capable to effect an immune deviation TH1 to TH2
and are therefore suitable for use against adjuvant arthritis. TH1 and
TH2 cells are CD4-expressing T cells. TH1 cells are also called
pro-inflammatory T helper cells and secern the cytokines IL-2, TNF and
IFN-γ. TH2 cells support the activation of B cells and secern the
cytokines IL-4, IL-5 and IL-10. The differentiation of CD4 T cells from
the above functionally different sub-groups is not only controlled by the
available cytokines, but it is also modulated by costimulation over CD28.
CD28-deficient mice show normal TH1, but reduced TH2-dependent answers
and the cytokine profile of TCR transgenic CD4 cells is displaced by CD28
ligation in the direction TH2. On the other hand, a strong TCR signal
will prevent CD28-mediated TH2 differentiation.

[0016]From the primary literature summarized in the document K. J. Maloy
et al., Nature Immunology, vol. 2, No. 9, pages 816 ff., 2001, it is
known that regulatory T cells are important for autoimmune reactions. For
instance in experimental animal models of the multiple sclerosis, of the
type 1 diabetes and of inflammatory intestinal diseases, the capability
of these cells to suppress the respective symptoms was shown.

[0017]The Guillain-Barre syndrome is an acute autoimmune-inflammatory
disease of the peripheral human nervous system. The incidence of GBS is 1
to 2 per 100,000 inhabitants. The chronic form is the chronic
demyelinating polyneuropathy (CDP). The incidence of CDP is 10 to 20 per
100,000 inhabitants. mAbs or related substances for the prevention and/or
treatment of these diseases are not known.

TECHNICAL OBJECT OF THE INVENTION

[0018]The invention is based on the technical object to specify a
pharmaceutical composition, by means of which regulatory T cells can be
stimulated and which is particularly suited for the prevention and/or
treatment of the multiple sclerosis, type 1 diabetes, inflammatory
intestinal diseases, GBS and/or CDP.

BASICS OF THE INVENTION AND PREFERRED EMBODIMENTS

[0019]For achieving the above technical object, the invention teaches the
use of a CD28-specific superagonistic monoclonal antibody (mAb) or of a
mimicry compound thereto, for making a pharmaceutical composition for the
induction and/or multiplication of regulatory T cells.

[0020]First of all, the invention is based on the finding that by means of
superagonistic CD28-specific substances, mAbs or mimicry compounds
hereto, CD4+CD25+ T cells can be induced, i.e. the number thereof is,
after treatment of an organism with the substance, distinctly higher than
in an organism that was not treated or was treated with
non-superagonistic substances.

[0021]Further, the invention is based on the finding that substances
according to the invention obviously are very good drugs for the
treatment of the Guillain-Barre syndrome and/or of the chronic
demyelinating polyneuropathy and other autoimmune-related diseases.
Therefore, the invention further teaches the use for treating these
diseases.

[0022]Superagonistic CD28-specific substances used according to the
invention, i.e. mAbs or mimicry compounds thereto, are those which
activate independently from the occupation of the antigen receptor
several to all sub-groups of the T lymphocytes.

[0023]The substance binds to CD28 or to a partial sequence thereof. The
partial sequence may for instance include an amino acid sequence Seq. ID
1 or 2-7 or 17, which lie at least partially in the region of the C'-D
loop of CD28. To one of the sequences with val at the 5' end, one or more
amino acids of the sequence 8 may be connected in the order defined
there. The loop is in the region with the sequence GNYSQQLQVYSKTGF.
Mimicry compounds according to the invention can be identified in a
screening method, a prospective mimicry compound or a mixture of
prospective mimicry compounds being subjected to a binding assay with
CD28 or a partial sequence herefrom, in particular the C'-D loop, and
substances binding to CD28 or to the partial sequence herefrom being
selected, possibly followed by an assay for testing for superagonistic
stimulation of several to all sub-groups of the T lymphocytes. In the
case of a mixture it will be suitable to perform a deconvolution. Among
the selected mimicry compounds so to speak a ranking according to the
selectivity and/or affinity may be established, highly affinitive
substances being preferred. In addition to or in lieu of such a ranking,
a ranking may be performed according to a quantification of the induction
of the regulatory T cells or according to the inhibition of the disease
for instance in an animal test by using disease models.

[0024]An example of a substance used according to the invention is a
superagonistic CD28-specific mAb. It can for instance be made by that a
non-human mammal is immunized with CD28 or a peptide comprising a partial
sequence herefrom, for instance as mentioned above or homologues hereto,
cells being taken from the non-human mammal cells and hybridoma cells
being produced from the cells, and the thus obtained hybridoma cells
being selected such that in their culture supernatant there are mAbs
binding to CD28. A humanization can be performed with conventional
methods. Suitable mAbs can alternatively be made by selecting B
lymphocytes binding to the loop, and by cloning their expressed
immunoglobulin genes. An isolation of suitable mAbs from phages libraries
is also possible.

[0025]In detail, this may be a mAb being obtainable from hybridoma cells,
as filed under the DSM numbers DSM ACC2531 (mAb: 9D7 or 9D7G3H11) or DSM
ACC2530 (mAb: 5.11A or 5.11A1C2H3). The mAb may comprise one or more of
the sequences Seq. ID 9, 11, 13 and/or 15, or one or more of the
sequences Seq. ID 10, 12, 14, 16, 18 and/or 19, or sequences being
homologous hereto or being (partially) coded thereby. In Seq. ID 13 the
nucleic acid sequence of the variable region of the heavy chain of a mAb
5.11A according to the invention is represented. Seq. ID 14 shows the
peptide coded thereby. Seq. ID 15 shows the nucleic acid sequence of the
variable region of the light chain of this mAb. Seq. ID 16 is the peptide
coded hereby. In Seq. ID 9 the nucleic acid sequence of the variable
region of the light chain of a mAb 9D7 according to the invention is
represented. Seq. ID 10 shows the peptide coded hereby. Seq. ID 11 shows
the nucleic acid sequence of the variable region of the heavy chain of
this mAb. Seq. ID 12 is the peptide coded hereby. Seq. ID 18 and 19 show
the amino acid sequences of the variable region of a humanized mAb 5.11A
of the light chain and of the heavy chain, respectively.

[0026]The invention finally also relates to treatments, wherein to a
person suffering from a disease caused by low regulator T cell counts or
high T lymphocytes infiltration in organs or tissues, for instance GBS
and/or CDP, a pharmaceutical composition according to the invention is
administered in a pharmacologically effective dose and in a galenic
preparation suitable for the administration.

[0027]In the following, the invention is explained in more detail, based
on examples representing embodiments only. Herein, in FIGS. 1 to 9 and
the text sections belonging hereto, methods and results are shown that
represent on the one hand target structures for finding suitable
substances and that describe on the other hand substances which can be
used according to the invention. In FIGS. 10 to 15 are represented
results that prove the induction of regulatory T cells by substances used
according to the invention. FIGS. 16 to 21 show results hat prove the
effect of substances according to the invention in an animal model, the
experimental allergic neuritis of the LEW rat (EAN). The EAN is a model
for the human GBS and the CDP (also called CIDP or chronic inflammatory
demyelinating poly-radiculoneuropathy). There are:

[0028]FIG. 1 the stimulation of T lymphocytes of the rat with different
CD28-specific mAbs (a: costimulation, b: superagonistic stimulation),

[0029]FIG. 2 a sequence comparison between mouse, rat and human CD28 in
the region of the C'-D loop (in box),

[0030]FIG. 3 experimental results for localizing the binding site of
superagonistic mAb at the CD28 molecule of the rat,

[0031]FIG. 4 the binding of different human CD28-specific mAbs at CD28 (a)
and costimulatory (b) and superagonistic (c) activity of the mAbs of FIG.
4a,

[0032]FIG. 5 binding tests that show that superagonistic mAbs specifically
bind to the C'-D loop,

[0033]FIG. 6 a three-dimensional representation of CD28 with marking of
the C'-D loop,

[0034]FIG. 7 experiments for the activation of cells by means of mAbs
according to the invention,

[0036]FIG. 9 the frequency of the CD4+CD25+ cells in the total population
of the CD4 cells of a rat, in comparison to after a treatment with a
superagonistic CD28-specific mAb and a costimulatory mAb,

[0037]FIG. 10 the phenotypic characterization of CD4+CD25+ cells induced
by superagonistic CD28-specific mAbs and the comparison with CD4+CD25-
and CD4+CD25+ cells from untreated control animals,

[0038]FIG. 11 the induction of the proliferation of CD4+CD25+ cells by
superagonistic CD28-specific mAbs in a cell culture,

[0039]FIG. 12 another phenotype of the CD4+CD25+ cells obtained according
to FIG. 11,

[0040]FIG. 13 the inhibitory function of the regulatory T cells,

[0041]FIG. 14 the experiments according to FIG. 11 with human T cells and
the use of superagonistic human-CD28-specific mAbs,

[0042]FIG. 15 the course of the active EAN disease under treatment with
superagonistic CD28-specific mAbs in comparison with costimulatory mAbs,

[0043]FIG. 16 the effect against ENA by administration of superagonistic
CD28-specific mAbs before the immunization with the autoantigen inducing
EAN,

[0044]FIG. 17 the treatment according to FIG. 15, however with a different
treatment plan,

[0045]FIG. 18 the treatment according to FIG. 15, however for the case of
the passive or adoptive transfer EAN,

[0046]FIG. 19 the sorting of human CD4+ cells in CD4+CD25+++ and CD4+CD25-
cells,

[0047]FIG. 20 the growth curves of T cells expanded by monoclonal
antibodies and IL-2 according to the invention of FIG. 19 (sorted),

[0048]FIG. 21 the CTLA-4 expression of the expanded T cells of FIG. 20,
and

[0049]FIG. 22 the functional analysis of the expanded T cells of FIG. 20
by means of a suppression assay, A: proliferation of the indicator cells
without and with stimulation (CD3/anti CD28), B: suppression of the
proliferation of the indicator cells in presence of expanded CD4+CD25+++
cells.

[0050]FIG. 1 shows the stimulation of freshly isolated T lymphocytes of
the rat in the form of a 3H thymidine incorporation. The method
corresponds to the one described in the document WO98/54225, to which
here and in the following explicitly reference is made and its disclosure
contents are herewith incorporated in the present text. In FIG. 1a is
shown the costimulation, i.e. T cell receptor (TCR) specific mAbs were
bound to the plastic surface in all wells. Because of lacking
costimulation, the negative control (uppermost bar) does not show any
incorporation. Costimulation is then given by the addition of
CD28-specific mAbs in a dissolved form. The complete shown range of
CD28-specific mAbs was used. This series of different CD28-specific mAbs
originates from an approach of the immunization and preparation of
hybridoma cell lines described in WO98/54225. These are culture
supernatants containing enough CD28-specific mAbs for a saturating
binding to 2105 T cells. From FIG. 1a can be taken that all of these
mAbs are able to activate in a costimulating manner, i.e. to excite the
thymidine incorporation in presence of the anti-TCR mAbs. In FIG. 1b is
shown the stimulation in absence of TCR-specific mAbs. This experiment,
too, was performed as described in WO98/54225. It can be seen that only
two mAbs are able to stimulate the T lymphocytes in absence of a TCR
signal. These mAbs have thus a superagonistic activity.

[0051]Further, it was investigated whether costimulatory and
superagonistic CD28-specific mAbs bind to different regions of the CD28
molecule. The mAbs were prepared by immunization of mice with CD28 of the
rat; as expected, they all do not react with mouse CD28 (not shown).
Since the mAbs can thus detect only such regions of the rat CD28 molecule
which are different from the mouse, first a sequence comparison between
the CD28 of the mouse and of the rat was made (see FIG. 2, upper
section). The differences between the two species are highlighted. For
identifying the amino acids, a one-letter code was used. As prototypes
for a conventional rat CD28-specific mAb JJ319 was used, for a
superagonistic mAb JJ316 was used (see WO98/54225).

[0052]In FIG. 3 is shown the mapping of the binding. Expression plasmids
were constructed, wherein a part of the extracellular domain of CD28
originates from the mouse, another one from the rat. This is symbolically
shown by bars or lines; on the right hand thereof is shown the binding of
the mAbs JJ316 and JJ319 to mouse fibroblasts (L929 cells) transfected
with these expression plasmids. In the first two lines of FIG. 3 (m/r and
r/m 1-37) the binding of the two antibodies to the "right-hand" half of
the sequence is mapped. Both bind, when the latter originates from the
rat. In the reversed construct (rm CD28 1-37, left hand rat, right hand
mouse) there is no binding. In the third line (m/r CD28 1-66) it is shown
that JJ316 does not bind anymore, whereas the still present part of the
rat sequences ("right-hand") sill suffices for the detection by JJ319.
According thereto, the two mAbs detect different epitopes on the CD28
molecule, and the binding of the superagonist JJ316 is therefore to be
searched in the region which originated in the construct of the first
line, not however in the construct of the third line from the rat. A
clear candidate here-for is the region in the box in FIG. 2.

[0053]In lines 4 and 5 of FIG. 3, therefore firstly two and then three
amino acids in this region of the mouse CD28 molecule were modified such
that they now represent the rat sequence. By this "transplantation" of
three amino acids only, the binding capability for mAb JJ316, not however
(as expected) that of JJ319 could be transferred. In Table 1 are
summarized the binding data for the complete range of CD28-specific mAbs.
There results a clear correlation: the two mAbs which function even
without TCR stimulation (superagonists) detect said epitope (in box in
FIG. 2), the conventional mAbs (only costimulatory) however do not. A
costimulatory mAb (5S35) detects the epitope in box very weakly and binds
very strongly to the "conventional" epitope.

[0054]The next two figures deal with superagonistic human-specific mAbs.
These, too, were prepared in mice, thus do not react with the CD28
molecule of the mouse. The mice were immunized with
human-CD28-transfected A20/J mouse B lymphoma cells (see WO98/54225) and
in addition boostered prior to the fusion with commercially available
human-CD28 FC fusion protein (bought from R and D Systems). In a series
of fusion experiments, from several thousand cell lines, approx. 20 were
identified producing human-CD28-specific mAbs (binding to mouse L929
cells expressing human-CD28, but not to untransfected L929 cells),
analogously to the screen in document WO98/54225. Two of these showed the
searched superagonistic activity (9D7 and 5.11A), whereas all new mAbs
have the conventional costimulatory activity. In the following, in
particular the two superagonistic mAbs are described. As an example for a
conventional human-CD28-specific mAb, the also newly generated mAb 7.3B6
was used.

[0055]FIG. 4a shows that the used preparations of the three new mAbs bind
comparatively well and also with a comparative titer to human T
lymphocytes. It is shown an experiment wherein freshly isolated
mononuclear cells from the human blood (so-called PBMC) firstly were
treated with different dilution steps of the used mAbs on ice; then they
were washed, and the bound mAb was made visible by a secondary antibody
marked with a fluorescence dye, said antibody specifically detecting the
bound mouse mAb. By the use of another mAb which detects human CD4 cells
and to which was bound a second fluorescence dye, the binding of the
titrated mAbs could be determined by electronic gating selectively for
the CD4 T lymphocytes. "MFI" is the median fluorescence intensity being a
measure for the amount of the bound CD28-specific mAb. The concentrations
are 1:3 dilutions of a standardized original preparation. It is fully
normal that in this test the highest concentration provides a weaker
signal than the following titration steps; this has to do with the
avidity (bivalent binding) of mAbs and does not play a role in the
contexts discussed here.

[0056]FIGS. 4b and c compare the capabilities of superagonistic
human-CD28-specific mAbs to those of conventional CD28-specific mAbs--in
presence and in absence of a TCR signal--to stimulate freshly isolated
human T cells to growth. Again a 3H thymidine incorporation is shown, as
described before for the rat. For FIG. 4b, the wells were coated with a
mAb reacting with the human TCR/CD3 complex. Thus costimulation was
measured. It can be seen that the proliferation does not occur without a
costimulation with one of the mAbs (negative control), all three
antibodies are however able to stimulate the cell division. For FIG. 4c,
the procedure took place in absence of a TCR/CD3-specific mAb. Only the
antibodies 9D7 and 5.11A could stimulate in a superagonistic manner.

[0057]After the epitope for superagonistic mAbs for the rat is defined,
and two new superagonistic mAbs with specificity for human CD28 have been
isolated, it was verified whether these mAbs bind to the corresponding
position of the human CD28 molecule. As can be seen in FIG. 2, the CD28
molecules of mouse and man differ in numerous positions. On the basis of
the mapping of the superagonistic epitope for the rat, it was therefore
directly verified whether the binding site for the superagonistic epitope
on human CD28 to the CD28 molecule of the mouse can be achieved by a
"transplantation" of the five amino acids of this homologous region. The
results are shown in FIG. 5. With the background of the homogeneously
represented mouse sequence for the extracellular domain of the CD28
molecule (center), the exchanged (mouse to human) amino acid positions
are shown as lines (bottom). The numbers at the sides in addition
indicate the individual positions and mutations (F60V means for instance
that at position 60 the phenylalanine of the mouse was replaced by a
valine of the human sequence). Moreover, the binding of the three
investigated mAbs is represented. As the figure shows, all three mAbs do
detect human CD28, however only the two mAbs 9D7 and 5.11A react with the
mouse CD28 molecule to which were transplanted the five amino acids of
the human CD28 at the decisive position. In view of the variety of
differences, this specific preparation of the reactivity is surprising
and confirms to a full extent the finding derived from the experiments
with rat CD28, namely that superagonistic mAbs must bind to a defined,
namely this position of the molecule.

[0058]FIG. 6 is a three-dimensional model of the CD28 molecule. The newly
defined binding region is highlighted. It corresponds to the sequence in
the box in FIG. 2. The extracellular domain of CD28 structurally belongs
to the immunoglobulin superfamily being characterized by two superimposed
P-pleated sheets as a basic structure. The labeling of these bands
follows a pattern given in the literature. It is important for the
representation shown here that the region identified as an epitope for
superagonistic CD28-specific mAbs in rat and mouse are designated "C'-D
loop". Thus it was shown that mAbs with specificity for the C'-D loop of
the CD28 molecule have superagonistic activity, that is, in the meaning
of the document WO98/54225, can be used for the activation of T
lymphocytes. The superagonistic activity of C'-D loop-specific mAbs in
rat and man shows that not the sequence of the epitope, but its position
or shape is important.

[0059]In the experiments of FIG. 7, it was investigated whether mAbs do
not only bind (see FIGS. 3 and 5), but whether there is really an
activation. For this purpose, T tumor cells of the mouse, BW, were
transfected either with the construct of FIG. 3, line 5 (rat C'-D loop
transfer) or with the construct of FIG. 5, line 3 (human C'-D loop). The
activation of these cells is not measured by cell division (they
proliferate anyway), but by the production of the cytokine IL-2. FIG. 7
shows that without stimulation there is no IL-2 production (negative
control). The stimulation with a T cell receptor-specific mAb induces
IL-2 production (positive control). FIG. 7a shows the results when using
the superagonistic mAb JJ316 of the rat, whereas FIG. 7b shows the
results for the human C'-D loop-specific mAb 5.11A. In either case the
respective cell lines are stimulated to IL-2 production. As expected, the
stimulation did however not take place by means of "conventional"
CD28-specific mAbs, since they do not only bind to the C'-D loop, but
cannot detect at all the construct, because they are specific for the rat
or human-specific sequences which are not included in the construct.

[0060]In FIG. 9 are shown dot plots, wherein every measured cell is
represented by a dot. The phenotypic characterization of the regulatory T
cells takes place by the combination of the cell surface molecules CD4
and CD25. For this purpose, the cell suspensions were incubated with
correspondingly fluorescence dye marked monoclonal antibodies against CD4
and CD25, washed and examined in a flow cytophotometer for the binding of
these antibodies. The shown results were obtained three days after IP
injection of a costimulatory (FIG. 9a, JJ319) or superagonistic
CD28-specific mAb (FIG. 9b, JJ316). In the case of JJ319, approx. 7% of
the CD4 T cells are also CD25- positive(4/(50+4)), which corresponds to
not shown results in untreated animals. However, after treatment with
JJ316, approx. 20% are CD4+CD25+ (10/(10+40)). Further, the level of the
CD25 expression is by far higher than in the control animal. Such a high
level is characteristic for regulatory T cells.

[0061]FIG. 10 shows a phenotypic characterization in a representation as a
histogram. In FIGS. 10a to 10c, the marker CD45RC was detected, a
high-molecular isoform of the CD45 molecule being expressed strongly on
naive CD4 T cells, however weakly on stimulated CD4 T cells. A weak
expression is typical for regulatory T cells. FIG. 10a shows that most
CD4+C25- cells from untreated animals strongly express CD45RC. However,
in the case of CD4+CD25+ cells from untreated animals, a strong
expression takes place in a clear minority of all cells (FIG. 10b). In
the case of the treatment with the superagonistic CD28-specific mAb (FIG.
10c), the downward regulation of CD45CD45RC is even more distinct than in
the case of FIG. 10b. In the case of FIGS. 10d to 10e, the CD152 (CTLA-4)
constitutively expressed by regulatory T cells is detected by staining.
This staining must be performed, because of the intracellular
localization of CD152, after permeabilization of fixed cells, and is
therefore provided with an unspecific background. For verifying this, a
so-called isotype control was performed, i.e. an intracellular staining
with a mAb of the same immunoglobulin class, which however cannot detect
anything specifically. The specific CD152 proof is obtained by a
displacement of the CD152 histogram with regard to the isotype control
histogram. In FIG. 10d cannot be seen a displacement and thus no CD152
expression in the CD4+CD25- cells. In the untreated CD4+CD25+ cells there
is a weak displacement (FIG. 10e) and in the JJ316 (superagonistic
CD28-specific mAb) treated cells there is a stronger displacement, in
agreement with the results of FIGS. 10a to 10c.

[0062]As a result, it is phenotypically shown, with FIGS. 9 and 10, how
regulatory cells can be identified, and that superagonistic CD28-specific
mAbs preferentially multiply or induce in vivo regulatory T cells.

[0063]In FIG. 11, CD4+CD25+ cells are isolated by electronic cell sorting
either from untreated rats (FIG. 11a) or from rats treated with JJ316
(FIG. 11b) and cultivated in 96 well plates according to the state of the
art. The cell multiplication was measured by 3H thymidine incorporation
between day 2 and 3 of the cultivation. "(-)" means no stimulation,
costimulation means stimulation with a non-superagonistic CD28-specific
mAb (JJ319) and with the TCR-specific R73, and JJ316 shows the
superagonistic stimulation. FIG. 11a as well as 11b show that regulatory
cells do not react well upon costimulation, however well upon stimulation
with a superagonistic CD28-specific mAb. Stimulation with mAbs used
according to the invention shows also in a cell culture a considerable
multiplication of regulatory T cells.

[0064]FIG. 13 shows a representation according to FIG. 10f, however after
in vitro stimulation with superagonistic CD28-specific mAbs (JJ316). An
even stronger detectable CD152 expression can be seen.

[0065]In FIG. 13 is shown the inhibitory function of regulatory T cells on
Cd4+CD25- T cells serving as indicator cells for the suppression effect.
As a stimulus for the CD4+CD25- T cells, costimulation (R73+JJ319) was
used. The 3H thymidine incorporation between day 2 and 3 of the
cultivation was measured. CD25+ means electronically sorted CD4+CD25+ T
cells from animals treated three days before with superagonistic
CD28-specific mAbs (JJ316). CD25- represents the indicator cells.
CD25+/CD25- means in FIG. 13a that both cell populations were mixed with
one another in identical parts. It can be seen that upon costimulation
the CD25+ cells do not react with proliferation. Further, the
proliferation of indicator cells is in addition suppressed. In FIG. 13b
is represented a titration of the regulatory T cells by a mixture with
indicator cells in different quantities. It can be seen that even with a
ratio of regulatory to indicator cells of 1:16, suppression can still be
observed. This shows the high effectivity of the regulatory cells
stimulated with mAbs used according to the invention.

[0066]FIG. 14 shows a comparison of the reactions of human CD4+CD25- T
cells (naive cells) to CD4+CD25+ T cells (regulatory cells) in response
to costimulation (anti-CD3+conventional anti-CD28 mAb) or to
human-specific superagonistic CD28-specific mAbs (9D7 and 5.11A). The
experiments correspond to those described above for the rat. Beginning at
the left-hand side, the first three groups show unstimulated controls (no
3H thymidine incorporation). These are all CD4+ T cells, then the CD25-
fraction gained by sorting and finally the CD25+ fraction gained by
sorting. "med" means medium. Then follow two groups wherein a
superagonistic stimulation was made (9D7 and 5.11A). It can be seen that
the regulatory T cells react in a better way on the stimulation with
superagonistic CD28-specific mAbs, compared to the total population of
the CD4+ cells and their CD25- fraction. The last group of three shows
the results of the conventional costimulation. Here, however, the
reaction of the unseparated T cells and of the CD25- fraction is rather
better.

[0067]As a result, it is proven for rat T cells as well as in the human
system that superagonistic CD28-specific mAbs induce or multiply
regulatory T cells in a better way than conventional costimulation.
Further, it is proven that this can also be verified in the intact
organism.

[0068]In FIG. 15 is represented the course of the active EAN disease under
treatment with various mAbs. 7-8 weeks old female LEW rats (obtainable
from Charles River Laboratories, Sulzfeld, Germany) were used. The
animals were immunized with a synthetic peptide corresponding to a part
of the myelin protein P2 wrapping peripheral nerve fibers (amino acids
53-78 of the bovine P2 protein, 50 μl of a 0.5 mg/ml solution,
inoculation in the foot balls). After approx. 10 days a progressive
paralysis develops which can be quantified according to a standardized
scoring (King et al., Exp. Neurol., 87:9-19 (1985)). FIG. 15a shows a
preparative therapy with superagonistic CD28-specific mAbs (JJ316) and
FIG. 15b a treatment with conventional mAbs (JJ319). The application took
place in 1 mg/animal doses IP either on the day of the P2 immunization
(D0), at day 12 (D12), i.e. after beginning of the symptoms, or on both
days. The various groups comprises 3 to 6 animals. A comparison of FIGS.
15a and 15b shows that the treatment with superagonistic CD28-specific is
distinctly more effective than the treatment with conventional mAbs.

[0069]FIG. 16 shows the results according to FIG. 15, however with a
prophylactic treatment. d-7 is a treatment 7 days before the
immunization, d-21 21 days before the immunization. It can be seen that a
resistant state can be achieved by multiplication of regulatory T cells
with a treatment within one week before the immunization, not however
with a treatment 3 weeks before the immunization.

[0070]FIG. 17 is based on an approach identical to FIG. 15, however
treatment with JJ316 at day 0 and 4. FIG. 17a shows the course of the
disease corresponding to the representation of FIG. 15a. FIG. 17b shows
electro-physical properties, namely speed of the stimulus transmission as
a direct clinical parameter for the damage of the sciatic nerve. The
measurements were performed according to the documents Adlkofer et al.,
Nat. Genet., 11:274-280 (1995) and Heininger et al., Ann. Neurol.,
19:44-49 (1986). The pathological results can be seen by means of control
groups in particular in the extension of the NI and F latencies (see days
0 and 12, open symbols). In contrast, the latencies in the case of the
animals treated with superagonistic CD28-specific mAbs (JJ316) remain
nearly unchanged between day 0 and day 12 (full symbols).

[0071]Not shown are supplementing examinations with a histological proof
of the T cell infiltration in thin layers of the nerves. The detection of
the cells took place with a mAb being suitable for this technology,
namely B115 and coloration of the T lymphocytes. The cell nuclei were
counter-colored in a different color. In comparative experiments it was
found that in the not treated control group, a higher number of T cells
were infiltrated than in the group treated with a superagonistic
CD28-specific mAb (JJ316), which indicates a suppression by regulatory T
cells by using mAbs according to the invention.

[0072]Further experiments are not shown wherein the isolating myelin
sheaths were colored. The treatment with superagonistic CD28-specific
mAbs showed a healthy picture, whereas the control group showed
demyelinization, i.e. destruction of the isolating sheaths.

[0073]In FIG. 18 are shown the results of a therapy of the passive or
active adoptive transfer EAN (AT-EAN). This is not induced by
immunization with the nerve antigen, as described above, but takes place
by intravenous injection of an autoreactive CD4+ T cell clone with
specificity for the P2 myelin antigen (8106, cell line G7) according
to the document Stienekemeier et al., Brain, 122:523-535 (1999). FIG. 18a
shows the course of the disease of a control group, under treatment with
JJ316 at day 1 (d1) and treatment at day 3 (d3). It is notable that even
after the beginning of the disease symptoms, i.e. treatment at day 3, the
disease can be stopped. In FIG. 18b the infiltration of the nerves with T
cells has been quantified for the three groups, and it can be seen that
in the control group this leads to a damage to the nerves. In contrast,
the T cell counts are considerably lower in case of a treatment with mAbs
used according to the invention because of the induction of regulatory T
cells.

[0074]In the experiments of FIGS. 19 to 22, it is finally proven that
CD4+CD25+++ T cells expanded by means of monoclonal antibodies according
to the invention can even after expansion maintain their functional
properties, e.g. the suppression of the proliferation of "conventional" T
cells. For this purpose, the CD4+ T cells from human peripheral
mononuclear cells (PBMC) were purified by means of magnetic separation
(negative depletion of CD8+, CD11b+, CD16+, CD19+, CD36+ and CD56+ cells;
purity 95%). These cells were then loaded with a CD28-specific antibody
and then with a PE-conjugated secondary antibody, sorted into CD4+CD25+++
and CD4+CD25- T cells (see FIG. 19) and cultivated after addition of
monoclonal antibodies according to the invention coupled to Dynabeads
(hulG4) and Interleukin 2 (IL-2) (day 0). On day 5, a coloration of the
expanded cells with anti-CD4 and anti-CD25 at the cell surface took
place, and intracellularly with anti-CTLA-4 and Ki-67. On day 6, the
separation of the beads from the cultivated cells and removal of the IL-2
by repeated washing was performed, followed by a 2-day culture in medium
alone. The two subpopulations multiplied tenfold within 8 days (see FIG.
20). The increased expression of the protein CTLA-4 in the expanded
CD4+CD25+++ cells (see FIG. 21) is an indication that these cells have
kept their regulatory phenotype. This was then verified as follows by a
functional characterization.

[0075]Syngeneic peripheral mononuclear cells from heparinized whole blood
were gained and marked with the fluorescence dye CFSE (carboxy
fluorescencein diacetate succinimidyl ester). These cells served as
indicators cells. Firstly, they were stimulated with anti-CD3 and
anti-CD28 antibodies for three days. With every cell division, the
intensity of the marking measurement values of these indicator cells were
halved (see FIG. 22a). In independent approaches, on day 8 expanded
CD4+CD25+++ or CD4+CD25- T cells, resp., were mixed with the CFSE-marked
indicator cells in a ratio 1:1 or 1:5, resp., and were cultivated
(stimulation with anti-CD3 mAb, clone HIT3a, final concentration 0.1
μg/ml and anti-CD28 mAb, clone CD28.2, final concentration 0.05
μg/ml). FIG. 22b shows that the number of cell divisions in the
indicator cells was strongly reduced by the presence of the expanded
CD4+CD25+++ T cells, whereas the CD4+CD25- T cells showed a weak effect
only. Thus it is proven that the regulatory CD4+CD25+++ T cells expanded
with monoclonal antibodies according to the invention were still able to
suppress the proliferation of other "normal" T cells.